Methods of treating ischemia with C2, N6 -disubstituted adenosine derivatives

ABSTRACT

This application relates to methods of treating myocardial or cerebral ischemia, comprising administering a compound of formula I: ##STR1## wherein X is halogen, perhalomethyl, cyano, C 1-6  -alkoxy, C 1-6  -alkylthio or C 1-6  -alkylamino; and 
     R 1  is selected from N-bonded heterocycles. 
     The compound with the greatest ability to discriminate between the A1 and A2 adenosine receptors is 2-chloro-N-[4-phenoxy-1-piperidinyl]adenosine.

This is a divisional application of application Ser. No. 07/963,878,filed, as PCT/DK91/00324 Oct. 24, 1991, now U.S. Pat. No. 5,432,164 thecontents of which are incorporated herein by reference in theirentirety.

The present invention relates to modified6-hydrazino-9-(β-D-ribofuranosyl)-(9H)-purines further substituted atthe 2-position and pharmaceutically acceptable addition salts thereofhaving certain very desirable central nervous system properties,processes for their preparation and their pharmaceutical compositions aswell as methods for using the compounds and compositions described.

BACKGROUND OF THE INVENTION

Adenosine can be considered to be a hormone which has been shown to havea number of significant effects on the mammalian central nervous system(CNS) [see, for example, Adenosine in the Nervous System (in the seriesNeuroscience Perspectives, Series Editor Jenner, P.) Stone, T. W., Ed.,Academic Press Ltd., London, 1991, Annual Reports in MedicinalChemistry, 1988, 23, 39-48; International Review of Neurobiology(Smythies, J. R. and Bradley, R. J., eds.) Academic Press Inc., 1985,27, 63-139.], especially under conditions of neuronal stress where thecompound appears to act as an endogenous neuro-protectant (Progress inNeurobiology, 1988, 31, 85-108, Trends in Pharmacological Sciences,1988, 9, 193-194). For example, the concentration of adenosine has beendemonstrated to rise greatly in certain brain regions followingepileptic seizures or conditions of neuronal ischaemia/anoxia, (BrainResearch 1990, 516, 248-256).

It has been established for some years now that centrally actingadenosine receptor agonists or compounds which increase extracellularadenosine levels can exhibit what is termed neuromodulator activity.Such substances influence the release of neurotransmitters in regions ofthe central nervous system (Annual Review of Neuroscience, 1985, 8,103-124; Trends in Neurosciences, 1984, 164-168), with particularinhibitory effects on the release of the excitatory amino acid glutamicacid (glutamate) (Nature, 1985, 316, 148-150, Journal of Neurochemistry,1992, 58, 1683-169).

There are several CNS ailments in which this adenosine receptor mediatedneuromodulator activity may be of clear therapeutic benefit. Examples ofthese would include the treatment of convulsive disorders (EuropeanJournal of Pharmacology, 1991, 195, 261-265; Journal of Pharmacology andExperimental Therapeutics, 1982, 220, 70-76), prevention ofneurodegeneration under conditions of brain anoxia/ischaemia(Neuroscience, 1989, 30, 451-462; Neuroscience Letters, 1987, 83,287-293; Medical Hypotheses, 1990, 32, 45-49, Pharmacology of CerebralIschaemia 1990 (Kriegelstein, J. and Oberpichler, H., Eds.,Wissenschaftliche Verlagsgesellschaft mbH: Stuttgart, 1990, pp 439-448)or the use of a purinergic agent in the treatment of pain (EuropeanJournal of Pharmacology, 1989, 162, 365-369; Neuroscience Letters, 1991,121, 267-270). The relevance of adenosine and adenosine agonists to allthese disease areas has recently been reviewed in Adenosine and AdenineNucleotides as Regulators of Cellular Function (Phyllis, J. W., Ed., CRCPress Inc: Boca Raton, Fla., 1991, pp 319-400).

Adenosine receptors represent a subclass (P₁) of the group of purinenucleotide and nucleoside receptors known as purinoreceptors. Thissubclass has been further classified into two distinct receptor typeswhich have become known as A1 and A2. Extensive research has beencarried out in a quest to identify selective ligands at these sites[see, for example, Comprehensive Medicinal Chemistry, Volume 3, (Hansch,C., Sammes, P. G. and Taylor, J. B., Pergamon Press PLC, 1990,601-642)].

Selective ligands exist for A1 and A2 adenosine receptors and thestructure-activity relationships of the various reference ligands havebeen reviewed (Biochemical Pharmacology, 1986, 35, 2467-2481) togetherwith their therapeutic potential (Journal of Medicinal Chemistry, 1992,35, 407-422). Among the known adenosine receptor agonists most selectivefor the A1 receptor over the A2 receptor are the examples where theadenine nucleus is substituted with a cycloalkyl group on the aminofunction, for example N-cyclopentyladenosine and N-cyclohexyladenosine(Journal of Medicinal Chemistry, 1985, 28, 1383-1384) or2-chloro-N-cyclopentyladenosine (Naunyn-Schmiedeberg's Arch. Pharmacol.1988, 337, 687-689).

Examples of adenosine derivatives in the chemical literature having anitrogen bonded directly to the 6-amino substituent are few in number,and are summarized below.

They include N-aminoadenosine, N-[(N-methyl-N-phenyl)amino]adenosine(Journal of Medicinal Chemistry, 1985, 28, 1636-1643);N-(methylamino)adenosine and N-[(N-hydroxy-N-methyl)amino]adenosine(Journal of Medicinal Chemistry, 1968, 11, 521-523);2-amino-N-aminoadenosine (Chemical and Pharmaceutical Bulletin, 1969,17, 2373-2376); 2-fluoro-N-aminoadenosine (Journal of MedicinalChemistry, 1970, 13, 427-430) and 2-fluoro-N-methoxyadenosine (Journalof Medicinal Chemistry, 1971, 13; 816-819). Finally, there is oneexample containing a cyclic amine, namely 2-amino-N-piperidinyladenosine(Arzneimittel-Forschung, 1970, 20, 1749-1751).

In the above scientific articles, no mention is made of anypharmacological effects of the compounds concerned on the centralnervous system.

In U.S. Pat. No. 3,819,613, substituted adenosine analogues withhydrazone derivatives on the 6-amino function are disclosed ashypotensive agents. In GB Patent No. 1,351,501, adenosine and2-aminoadenosine derivatives having a --NH--R₂ group joined to the6-amino function are disclosed as coronary dilators and plateletaggregation inhibitors. In EP Publication No. 152,944A, a series of 2-,6- and 8- substituted adenosine derivatives are described havingactivity as anti-allergy agents. In EP Publication No. 253,962A,adenosine and 2-haloadenosine analogues having an alkyl, cycloalkyl oran aralkyl group attached to the 6-amino function are described withactivity as antidementia agents.

In EP Publication No. 402,752A, derivatives of adenosine unsubstitutedin the 2-position are described which have a substituted heteroaromatic1-pyrrolyl moiety attached to the 6-amino group. In PCT Publication No.WO 91/04032, methods of preventing neural tissue damage inneuro-degenerative diseases by increasing extracellular concentrationsof adenosine are described. Examples are given of prodrug esters of AICAriboside which are claimed to be centrally acting neuroprotectiveagents. In PCT Publication No. WO 92/02214, analogues of AICA ribosideare described which increase extracellular adenosine levels withbeneficial effects claimed in peripheral and CNS ischaemia. In PCTPublication No. WO 90/05526, 2-(alkylalkynyl)adenosine derivatives aredescribed for treatment of ischaemic disease of the heart and brain. InEP Publication No. 0 423 777 A2 a method fur treating gastrointestinalmotility disorders using N(6) (substituted aminoalkyl) adenosinederivatives is disclosed. EP Publication No. 0 490 818 A1 describes anew use of 2'-O-methyl adenosine derivatives for a range of ailmentsincluding neurodegenerative disorders.

The present invention relates to new adenosine analogues havingremarkably potent binding in vitro to the adenosine A1 receptor and atthe same time showing selectivity for A1 receptor binding in vitro overthat of the A2 receptor subtype. In addition, the compounds contained inthis invention have a relatively high lipophilicity, especially whencompared to adenosine analogues which are not substituted on the 6-aminogroup or the purine 2-position. This latter property makes thesecompounds suitable for passage across the blood brain barrier, andsupports the suggestion that the compounds may be candidate drugs forthe CNS ailments mentioned within this invention.

The possibility that some of the compounds may be substrates fornucleoside-specific active transport systems across the blood barrieris, however, not excluded. These useful properties support thesuggestion that the compounds may be candidate drugs for the CNSailments mentioned above in humans. There are instances where it hasbeen demonstrated that co-administration of a peripherally activeadenosine receptor antagonist can lower the expected side effects on thecardiovascular system when an adenosine agonist is used as aneuroprotectant in animal models (Journal of Molecular Neuroscience,1990, 2, 53-59). This method of lowering side-effects is also applicableduring the therapeutic use of the adenosine receptor agonists covered bythe present invention.

The novel compounds of the invention are purine deriratives of formula(I), or a pharmaceutically acceptable salt thereof: ##STR2## wherein

X is halogen, perhalomethyl, cyano, C₁₋₆ -alkoxy, C₁₋₆ -alkylthio orC₁₋₆ -alkylamino;

R¹ is selected from the groups consisting of ##STR3## wherein n is 1 to3 and where the group (a) may be optionally substituted with one or twoC₁₋₆ -alkyl groups, C₂₋₆ -alkenyl, C₂₋₆ -alkynyl, phenoxy,phenylsulphonyl, phenylthio, hydroxy, phenyl, C₁₋₆ -alkoxy or C₁₋₆-alkoxy-C₁₋₆ -alkyl, phenylthioalkyl or ##STR4## wherein Y is O, S orNZ, where Z is H, C₁₋₆ -alkyl or phenyl, and where the group (b) may beoptionally substituted with C₁₋₆ -alkyl, C₂₋₆ -alkenyl, C₂₋₆ -alkynyl,phenoxy, phenyl, C₁₋₆ -alkoxy or C₁₋₆ -alkoxy-C₁₋₆ -alkyl, or ##STR5##which may be optionally substituted with C₁₋₆ -alkyl, C₂₋₆ -alkenyl,C₂₋₆ -alkynyl, phenoxy, phenylthio, phenyl, C₁₋₆ -alkoxy or C₁₋₆-alkoxy-C₁₋₆ -alkyl.

In certain examples, the group R¹ can contain one or more asymmetriccarbon atoms in addition to those asymmetric centres already present inthe molecule. In examples where this is the case, this inventionincludes all resulting diastereoisomers and mixtures thereof.

Various salts of compounds of formula (I) can be prepared which can beconsidered physiologically acceptable. These include addition saltsderived from inorganic or organic acids, for example, acetates,fumarates, glutarates, glutaconates, lactates, maleates,methanesulphonates, phosphates, salicylates, succinates, sulphates,sulphamates, tartrates and paratoluenesulphonates. In some cases,solvates of either the free nucleosides or the acid addition salts canbe isolated and these solvates may, for example, be hydrates oralcoholates.

Compounds of formula (I), which act as adenosine receptor agonists, arefound to be useful in the treatment of central nervous system conditionssuch as anxiety, neuronal ischaemia/anoxia, convulsive disorders(epilepsy) and neurodegeneration (including Parkinson's disease). Thisincludes treating disorders where the blood flow to the brain isinterrupted, for example during traumatic head injury, cardiac arrestand stroke.

Further, the compounds of formula (I) are found to be useful asanalgesic agents, in lowering plasma FFA levels or as cardiovascularagents.

The invention also relates to methods of preparing the above mentionedcompounds. These methods comprise:

Method A

A compound of formula (I) may be prepared by reacting a substance offormula (II), wherein L represents a leaving group such as a halogenatom (e.g. a chlorine or bromine, atom) or a trimethylsilyloxy group, R²and R³ are the same or different and represent hydrogen or a protectinggroup such as benzoyl-, p-toluoyl-, lower alkanoyl- (e.g. acetyl-), a2,3-O-(1-methyl)ethylidene group or a substituted silyl group (e.g. atrimethylsilyl or t-butyldimethylsilyl group) with a hydrazinederivative of general formula (III) ##STR6## giving the compound offormula (IV) as the reaction product. In cases where R² and R³ are nothydrogen an additional step will be required to remove protecting groupsfrom (IV); in cases where the groups R² and R³ are for example acetyl orbenzoyl, suitable conditions for deprotection include methanolicammonia, an alkali metal carbonate in methanol, an alkali metal alkoxidein the corresponding alcohol. Where the protecting groups are forexample alkylsilicon or arylsilicon derivatives, suitable deprotectionmethods include for example treatment with tetraalkylammonium fluoridesor aqueous hydrolysis in the presence of acid or base.

Method B

A compound of formula (I) (wherein X represents --NH--R⁴, or --O--R⁴,where R⁴ is C₁₋₆ -alkyl) may be prepared by reacting a substance ofgeneral formula (V) ##STR7## (where L is a leaving group as defined inmethod (A)) with a nucleophile, for example C₁₋₆ -alkylamino (optionallyin the presence of a suitable base) or with the anion (C₁₋₆ -alkoxide orC₁₋₆ -thioalkoxide) to afford the product (IV). In cases where R² and R³are hydrogen, compound (I) can be obtained directly. However, in caseswhere R² and R³ are not hydrogen an additional step will be involved toremove protecting groups from (IV); examples of conditions for removalof protecting groups are given in process (A). In some reactionsinvolving (V) with the anion (C₁₋₆ -alkoxide or C₁₋₆ -thioalkoxide),where R² and R³ are for example acetyl- or benzoyl-, partial or fulldeprotection may take place. In cases where only partial, deprotectionhas taken place, deprotection can be completed under conditionsexemplified in method (A).

Method C

A compound of formula (I) may be prepared by reacting a substance of thegeneral formula (VI) (where B represents --NH--R¹ or L as definedpreviously) with a diazotizing agent (such as, for example,3-methylbutyl nitrite) to form an intermediate species which can bereacted further with a variety of substrates as exemplified below inorder to introduce the group -X into the product (VII). ##STR8##

In the case where B represents a leaving group L, a further displacementreaction with for example (III) will be required in order to obtain theproduct (IV). In cases where the groups R² and R³ are not hydrogen, ornot all hydrogen, another step will be required to remove protectinggroups from (IV); conditions for removing protecting groups aredescribed in method A.

Methods for assessing adenosine receptor binding in vitro have beenreviewed [Adenosine Receptors, (Cooper, D. M. F. and Londos, C., eds.)Alan R. Liss, Inc., New York, 1988, 43-62].

Evaluation of these compounds in established animal models has indicatedthat the compounds according to the invention possess desirable centralnervous system properties. For example, they act as anticonvulsantagents, are effective in animal models of pain, and showcerebro-protective effects in laboratory test animals subjected tosimulated cerebral ischaemia. In addition, the compounds may haveefficacy as neuroprotective agents in cases of cerebral oedema andtraumatic head injury.

Evaluation of in vitro binding to adenosine A1 and A2 receptors

The affinity of the novel compounds described in this invention for theadenosine A1 receptor was determined essentially as described in theliterature using [³ H]-L-PIA as a radioligand (Naunyn-Schmiedeberg'sArchives of Pharmacology, 1980, 313, 179-187). Affinity for the A2receptor was measured using the radioligand [³ H]-CGS 21680 (EuropeanJournal of Pharmacology, 1989, 168, 243-246), and the values forrepresentative compounds is given in the table below. In vitro receptorbinding values obtained for the reference standards CFA[N-(cyclopentyl)adenosine] and L-PIA [N-(1-phenyl-2-propyl)adenosine]are included for comparison.

DMCM INDUCED SEIZURES IN MICE, I. P. 30 min.

Method description

DMCM, 15 mg/kg, i.p., clonic convulsions

RATIONALE

DMCM is an inverse agonist at the benzodiazepine receptor, presumablyproducing seizures by decreasing the potency of inhibition of the GABAreceptor/benzodiazepine receptor/chloride ionophore complex (1).

METHODS

15 mg/kg of DMCM dissolved in 0.02 N HCl (1 mg/ml) is administered i.p.in a volume of 300 μl to male NMRI mice weighing 20±2 g. This inducestwo different responses: a) some animals manifest a brief loss ofrighting reflexes or take up an upright position in which they have amild short clonus of the upper extremities, b) other animals manifestintense clonic and tonic convulsions of all extremities often followedby death. DMCM is administered 30 min. after an intraperitonealinjection of a test compound. The latency time for the presence ofintense clonic and tonic convulsions and death is noted until 15 min.after administration of DMCM. At least 5 doses of each test compound aretested with 8 mice per dose.

RESULTS

An anticonvulsive ED₅₀ value is determined as the dose (mg/kg)protecting 50% of the animals against clonic convulsions. This method isdescribed in more detail in Eur. J. Pharmacol. 94, 117-124, 1983.

The results obtained by testing compounds disclosed in the presentinvention are shown in the following table I.

                  TABLE I                                                         ______________________________________                                                 A1       A2              DMCM-ind.                                   Adenosine                                                                              Receptor Receptor        seizures                                    agonist  Binding  Binding   Ratio (ED.sub.50,                                 tested   (Ki, nM) (Ki, nM)  A2/A1 mg/kg)                                      ______________________________________                                        Example 5                                                                              4        691       173   0.1                                         Example 9                                                                              4        1143      289   0.7                                         Example 16                                                                             11       1733      158   1.0                                         Example 17                                                                             1.4      1200      857   0.9                                         CPA      1.6      173       108   0.2                                         L-PIA    2        134       67    0.1                                         ______________________________________                                    

The compounds of the invention, together with a conventional adjuvant,carrier, or diluent, and if desired in the form of a pharmaceuticallyacceptable acid addition salt thereof, may be placed into the form ofpharmaceutical compositions and unit dosages thereof, and in such formmay be employed as solids, such as tablets of filled capsules, orliquids, such as solutions, suspensions, emulsions, elixirs, or capsulesfilled with the same, all for oral use, in the form of suppositories forrectal administration; or in the form of sterile injectable solutionsfor parenteral use (including subcutaneous administration and infusion).Such pharmaceutical compositions and unit dosage forms thereof maycomprise conventional ingredients in conventional proportions, with orwithout additional active compounds or principles, and such unit dosageforms may contain any suitable effective amount of the adenosinereceptor agonist commensurate with the intended daily dosage range to beemployed. Tablets containing ten (10) milligrams of active ingredientor, more broadly, ten (10) to hundred (100) milligrams, per tablet, areaccordingly suitable representative unit dosage forms.

The compounds of this invention can thus be used for the formulation ofpharmaceutical preparation, e.g. for oral and parenteral administrationto mammals including humans, in accordance with conventional methods ofgalenic pharmacy.

Conventional excipients are such pharmaceutically acceptable organic orinorganic carrier substances suitable for parenteral or enteralapplication which do not deleteriously react with the active compounds.

Examples of such carriers are water, salt solutions, alcohols,polyethylene glycols, polyhyroxyethoxylated castor oil, gelatine,lactose, amylose, magnesium stearate, talc, silicic acid, fatty acidmonoglycerides and diglycerides, pentaerythritol fatty acid esters,hydroxymethylcellulose and polyvinylpyrrolidone.

The pharmaceutical preparations can be sterilized and mixed, if desired,with auxiliary agents, emulsifiers, salt for influencing osmoticpressure, buffers and/or colouring substances and the like, which do notdeleteriously react with the active compounds.

For parenteral application, particularly suitable are injectablesolutions or suspensions, preferably aqueous solutions with the activecompound dissolved in polyhydroxylated castor oil.

Ampoules are convenient unit dosage forms.

Tablets, dragees, or capsules having talc and/or a carbohydrate carrieror binder or the like, the carrier preferably being lactose and/or cornstarch and/or potato starch, are particularly suitable for oralapplication. A syrup, elixir or the like can be used in cases where asweetened vehicle can be employed.

Generally, the compounds of this invention are dispensed in unit formcomprising 0.05-100 mg in a pharmaceutically acceptable carrier per unitdosage.

The dosage of the compounds according to this invention is 0.1-300mg/day, preferably 10-100 mg/day, when administered to patients, e.g.humans, as a drug.

A typical tablet which may be prepared by conventional tablettingtechniques contains:

    ______________________________________                                        Active compound  5.0       mg                                                 Lactosum         67.0      mg Ph.Eur.                                         Avicel ™      31.4      mg                                                 Amberlite ™ IRP 88                                                                          1.0       mg                                                 Magnesii stearas 0.25      mg Ph.Eur.                                         ______________________________________                                    

As a result of their activity against pain or convulsive disorders andprevention of neurodegeneration under conditions of anoxia/ischaemia thecompounds of the invention are extremely useful in the treatment ofrelated symptoms in mammals, when administered in an amount effectivefor agonist activity of compounds of the invention. The compounds of theinvention may accordingly be administered to a subject, e.g., a livinganimal body, including a human, in need of adenosine receptor agonist,and if desired in the form of a pharmaceutically acceptable acidaddition salt thereof (such as the hydrobromide, hydrochloride, orsulphate, in any event prepared in the usual or conventional manner,e.g., evaporation to dryness of the free base in solution together withthe acid), ordinarily concurrently, simultaneously, or together with apharmaceutically acceptable carrier or diluent, especially andpreferably in the form of a pharmaceutical composition thereof, whetherby oral, rectal, or parenteral (including subcutaneous) route, in aneffective amount of adenosine receptor agonist, and in any event anamount which is effective for the treatment of anoxia, traumatic injury,ischaemia, migraine or other pain symptoms, epilepsy, orneurodegenerative diseases owing to their adenosine receptor agonistactivity. Suitable dosage ranges are 1-200 milligrams daily, 10-100milligrams daily, and especially 30-70 milligrams daily, depending asusual upon the exact mode of administration, form in which administered,the indication toward which the administration is directed, the subjectinvolved and the body weight of the subject involved, and the preferenceand experience of the physician or veterinarian in charge.

The preparation of compounds of formula (I) and preparations containingthem is further illustrated in the following examples.

Hereinafter, TLC is thin layer chromatography, THF is tetrahydrofuran,TFA is trifluoracetic acid and m.p. is melting point. Where meltingpoints are given, these are uncorrected. The structures of the compoundsare confirmed by assignment of NMR spectra (from which representativepeaks are quoted) and by microanalysis where appropriate. Compounds usedas starting materials are either known compounds or compounds which canbe prepared by methods known per se. Column chromatography was carriedout using the technique described by Still, W. C. et al., Journal ofOrganic Chemistry, 1978, 43, 2923 on Merck silica gel 60 (Art 9385).HPLC was carried out on a Waters model 510 chromatograph interfaced viaa system module to a Waters 490 multiwavelength detector to a reversedphase C₁₈ column (250×4 mm, 5 μm, 100 Å; eluent flow rate 1 mL/min. at35° C.). Retention times are given in minutes.

EXAMPLE 1 (METHOD A) 2-Chloro-N-(4-morpholinyl)adenosine

2,6-Dichloro-9(H)-purine (5.8 g, 30.7 mmol) and1-0-acetyl-2,3,5-tri-O-benzoyl-D-ribofuranose (16.26 g, 32.2 mmol) werethoroughly mixed and fused together at 145°-150° C. under oil pumpvacuum. The resultant gummy mixture was stirred gently for 0.75 hours(during which time the acetic acid by-product evaporated), cooled to ca.50° C. and dissolved in dichloromethane (100 ml) with stirring. Thissolution was applied directly to a column of silica gel (6×22 cm) andeluted initially with cyclohexane/dichloromethane (1/1), then withdichloromethane and finally with cyclohexane/ethyl acetate (1/1) toprovide 2,6-dichloro-2,3,5-tri-O-benzoyl-β-D-ribofuranosyl-(9H)-purine(16.6 g, 87%) as a colourless foam, TLC r_(f) 0.50 [SiO₂,cyclohexane/ethyl acetate (1/1)]. ¹ H NMR (400 MHz, CDCl₃) δ 4.72 (1H,dd, H-5'_(a)), 4.88 (1H, q, H-4'), 4.93 (1H, dd, H-5'_(b)), 6.15 (2H, m,H-2' & H-3'), 6.50 (1H, d, H-1'), 7.34-7.65 (9H, m, m- & p-ArH),7.90-8.13 (6H, m, o-ArH), 8.28 (1H, s, H-8). (This method of preparationis similar to the one described by Imai, K-i. et al., Chemical andPharmaceutical Bulletin, 1966, 14, 1377-1381, but without the use of acatalyst).

The above 2,6-dichloro-2,3,5-tri-O-benzoyl-β-D-ribofuranosyl-(9H)-purine(1.26 g, 2 mmol) and 4-aminomorpholine (0.89 g, 8.8 mmol) were dissolvedin a mixture of dioxane (30 ml) and toluene (15 ml). The solution washeated at 50° C. for 20 hours after which time it was confirmed that thestarting material was consumed and a new product was observed with TLCr_(f) 0.20 [SiO₂, ethyl acetate/dichloromethane (4/1)]. The cooledreaction mixture was evaporated to a gum and coevaporated with methanol(20 ml). Dried potassium carbonate (0.55 g, 4 mmol) and methanol wereadded and stirring was continued for 20 hours, whereupon acetic acid(1.0 ml) was introduced. The reaction mixture was evaporated and theresidue was coevaporated with toluene (30 ml) before purification bycolumn chromatography on silica gel (2.5×20 cm). Elution withdichloromethane initially, gradually increasing the polarity of theeluent to a mixture of dichloromethane/ethanol/25% aqueous ammoniasolution (100/10/1) provided the title compound (0.43 g, 55%) assemi-solid foam, TLC r_(f) 0.24 [SiO₂, dichloromethane/ethanol/25%aqueous ammonia solution (60/10/1)], ¹ H NMR (400 MHz, Me₂ SO-d₆) δ 3.56(1H, m, H-5'a), 3.67 (1H, m, H-5'b), 3.95 (1H, q, H-4'), 4.14 (1H, m,H-3'), 4.51 (1H, q, H-2'), 5.84 (1H, d, H-1'), 8.43 (1H, s, H-8). Thisnucleoside could be recrystallised from ethyl acetate/trace ethanol toprovide an analytical sample (0.3 g) as a white solid, m.p. 160°-161° C.(after drying in vacuo).

C₁₄ H₁₉ N₆ ClO₅. H₂ O requires C, 41.5; H, 5.2; N, 20.75; Cl, 8.75%.Found: C, 41.7; H, 5.35; N, 20.3; Cl, 8.6%.

EXAMPLE 2 2-Chloro-N-[1-(2,3,4,5,6,7-hexahydro)azepinyl]adenosine

The title compound was prepared according to method A as described inExample 1 and obtained as a foam (0.12 g, 62% from2,6-dichloro-2,3,5-tri-O-benzoyl-β-D-ribofuranosyl-(9H)-purine); ¹ H NMR(400 MHz, Me₂ SO-d₆) δ 3.55 (2H, 2m, H-5' and H-5'_(b)), 3.95 (1H, q,H-4'), 4.12 (1H, m, H-3'), 4.50 (1H, q, H-2'), 5.82 (1H, d, H-1'), 8.38(1H, s, H-8). HPLC retention time 18.39 (gradient elution, 15-35%acentonitrile/0.1M pH 3.3 ammonium sulphate buffer: 214 nm detector);99.9% purity.

EXAMPLE 3 2-Chloro-N-(2,6-dimethyl-1-piperidinyl)adenosine

The title compound was prepared according to method A as described inExample 1 and obtained as a foam (a mixture of diastereoisomers) (0.63g, 61% from2,6-dichloro-2,3,5-tri-O-benzoyl-β-D-ribofuranosyl-(9H)-purine); ¹ H NMR(400 MHz, Me₂ SO-d₆) δ 3.55 (1H, m, H-5'_(a)), 3.65 (1H, m, H-5'_(b)),3.95 (1H, m, H-4'), 4.12 (1H, m, H-3'), 4.55 (1H, q, H-2'), 5.82 (1H,2d, H-1'), 8.35, 8.40 (1H, 2s, H-8). HPLC retention time 19.61 (gradientelution, 15-35% acetonitrile/0.1M pH 3.3 ammonium sulphate buffer: 214nm detector).

EXAMPLE 4 2-Chloro-N-(4-methyl-1-piperazinyl)adenosine

The title compound was prepared according to method A as described inExample 1 and obtained as a foam (0.2 g, 14% from2,6-dichloro-2,3,5-tri-O-benzoyl-β-D-ribofuranosyl-(9H)-purine); ¹ H NMR(400 MHz, Me₂ SO-d₆) δ 3.56 (1H, m, H-5'_(a)), 3.67 (1H, m, H-5'_(b)),3.96 (1H, m, H-4'), 4.13 (1H, m, H-3'), 4.50 (1H, m, H-2'), 5.84 (1H, d,H-1'), 8.40 (1H, s, H-8). HPLC retention time 10.00 (gradient elution,15-35% acetonitrile/0.1M pH 3.3 ammonium sulphate buffer: 214 nmdetector).

EXAMPLE 5 2-Chloro-N-(1-piperidinyl)adenosine

2,6-Dichloro-2,3,5-tri-O-benzoyl-β-D-ribofuranosyl-(9H)-purine (20.0 g,31.7 mmol) (prepared as described in example 1), 1-aminopiperidine (6.35g, 63.4 mmol) and N,N-diisopropylethylamine (8.20 g, 63.4 mmol) weredissolved in dioxane (300 ml), and after 2.5 hours TLC indicated thatthe starting material was consumed. Dichloromethane (500 ml) was addedand the mixture was washed with water (2×150 ml). The organic phase wasdried (MgSO₄) and evaporated in vacuo to a foam. The foam was treatedwith methanol (120 ml) (causing crystallisation) and the vessel was keptat -10° C. for 1 hour. The product,2',3',5'-tri-O-benzoyl-2-chloro-N-(1-piperidinyl)adenosine, wascollected as white crystals (19.4 g, 88%), m.p. 110°-112° C.; ¹ H NMR(400 MHz, Me₂ SO-d₆) δ 4.68 (1H, dd, H-5'_(a)), 4.80 (1H, dd, H-5'_(b)),4.88 (1H, q, H-4'), 6.20 (1H, t, H-3'), 6.50 (1H, d, H-1'), 6.85 (1H, t,H-2'), 8.45 (1H, s, H-8).

C₃₆ H₃₃ N₆ ClO₇. H₂ O requires C, 60.45; H, 4.9; N, 11.75%. Found: C,60.45; H, 4.8; N, 11.3%.

The above 2',3',5'-tri-O-benzoyl-2-chloro-N-(1-piperidinyl)adenosine(19.2 g, 27.5 mmol) was dissolved in methanolic ammonia (150 ml)(previously saturated at -10° C.) and stirred at room temperature for 18hours. The precipitated benzamide was removed by filtration, and thefiltrate was evaporated to a fawn oil, which was triturated with diethylether to provide the title compound (6.0 g, 57%) as a white foam, ¹ HNMR (400 MHz, Me₂ SO-d₆) δ 3.55 (1H, m, H-5'_(a)), 3.66 (1H, m,H-5'_(b)), 3.94 (1H, q, H-4'), 4.12 (1H, m, H-3'), 4.50 (1H, q, H-2'),5.82 (1H, d, H-1'), 8.40 (1H, s, H-8); HPLC retention time 26.57(gradient elution, 5-25% acetonitrile/0.1M pH 3.3 ammonium sulphatebuffer: 214 nm detector); purity 99%.

EXAMPLE 6 2-Chloro-N-(2-phenyl-1-piperidinyl)adenosine

The title compound was prepared according to method A as described inExample 1 was obtained as a solid (0.25 g, 26%) by the reaction of1-amino-2-phenylpiperidine (Overberger, C. G. and Herin, L. P. Journalof Organic Chemistry, 1962, 27, 417) with9-(2',3',5'-tri-O-benzoyl-β-D-ribofuranosyl)-2,6-dichloro-9H-purine,followed by deprotection (as described in Example 5) to give the titlenucleoside (a ca. 60:40 mixture of diastereoisomers): m.p. 186°-210° C.;¹ H NMR (400 MHz, Me₂ SO-d₆) δ 3.47-3.20 (2H, m, 5'-CH₂), 3.82-3.97,4.04-4.14 (2H, 2m, H-3' and H-4'), 4.46-4.56 (1H, 2q, H-2'), 5.33, 5.73(1H, 2d, H-1'), 8.26, 8.48 (1H, 28, H-8).

EXAMPLE 7 (R)-2-Chloro-N-[2-(methoxymethyl)-1-pyrrolidinyl]adenosine

The title compound was prepared as described in Example 5 and obtainedas a semi-solid foam (0.49 g, 37% from9-(2',3',5'-tri-O-benzoyl-β-D-ribofuranosyl)-2,6-dichloro-9H-purine); ¹H NMR (400 MHz, Me₂ SO-d₆) δ 3.56 (1H, m, H-5'_(a)), 3.67 (1H, m,H-5'_(b)), 3.95 (1H, q, H-4'), 4.13 (1H, 1, H-3'), 4.53 (1H, q, H-2'),5.82 (1H, d, H-1'), 8.40 (1H, s, H-8); HPLC retention time 5.6 (gradientelution, 20-80% acetonitrile/water (containing 0.1% TFA): 250 nmdetector); purity 99%.

EXAMPLE 8 (S)-2-Chloro-N-[2-(methoxymethyl)-1-pyrrolidinyl]adenosine

The title compound was prepared as described in Example 5 and obtainedas a foam (0.66 g, 50% from9-(2',3',5'-tri-O-benzoyl-β-D-ribofuranosyl)-2,6-dichloro-9H-purine); ¹H NMR (400 MHz, Me₂ SO-d₆) δ 3.56 (1H, m, H-5'_(a)), 3.67 (1H, m,H-5'_(b)), 3.95 (1H, q, H-4'), 4.13 (1H, q, H-3'), 4.53 (1H, q, H-2'),5.82 (1H, d, H-1'), 8.40 (1H, s, H-8); HPLC retention time 5.6 (gradientelution, 20-80% acetonitrile/water (containing 0.1% TFA): 250 nmdetector); purity 98%.

EXAMPLE 9 (Method C) 2-Fluoro-N-(1-piperidinyl)adenosine

9-(2',3',5'-Tri-O-acetyl-β-D-ribofuranosyl)-2-amino-6-chloro-(9H)-purine(6.0 g, 14 mmol) (prepared as described by Robins, M. J. and Uznanski,B., in Nucleic Acid Chemistry (Townsend, L. B. and Tipson, R. S., eds.,)John Wiley and Sons, Inc., 1986, 3, 144) was dissolved in dioxan (100ml). 1-Aminopiperidine (1.83 ml, 16.95 mmol) was added followed bytriethylamine (2.33 ml, 18.5 mmol) and the solution was stirred for 190hours at room temperature. The reaction mixture was filtered, evaporatedand the resultant residue was purified by column chromatography onsilica gel eluting with ethyl acetate/cyclobexane (3/1) to afford2',3',5'-tri-O-acetyl-2-amino-N-(1-piperidinyl)adenosine (4.88 g, 71%)as a foam which solidified on coevaporation with methanol: m.p. 77°-790°C. ¹ H NMR (400 MHz, Me₂ SO-d₆) δ 2.04 (6H, s, 2' and 3'-O-acetyl-CH₃),2.13 (3H, s, 5'-O-acetyl-CH₃), 4.30 (2H, m, H-5'_(a) and H-4'), 4.40(1H, dd, H-5'_(b)), 6.03 (1H, d, H-1').

The above 2',3',5'-tri-O-acetyl-N-(1-piperidinyl)adenosine (1.33 g, 2.7mmol) was dissolved in THF (50 ml) and the temperature of the solutionwas held between -10° C. and -20° C. Fluoroboric acid (48%, 100 ml) wasadded followed by an aqueous solution of sodium nitrite (0.75 g/ml, 1.5ml). Addition of three identical amounts of sodium nitrite was continuedat 0.3 hour intervals, after which TLC investigation (on a neutralisedsample) indicated that the starting material was consumed [r_(f) 0.47(SiO₂, ethyl acetate/methanol (90/10))]. The pH of the reaction mixturewas adjusted to ca. 6 with 50% sodium hydroxide solution with cooling tobelow 0° C. and the now red reaction mixture was diluted with water (250ml). The solution was extracted with chloroform (2×100 ml) and thecombined extracts were dried (MgSO₄). The residue on evaporation(containing 2',3',5'-tri-O-acetyl-2-fluoro-N-(1-piperidinyl)adenosinewas treated with methanolic ammonia (150 ml) (previously saturated at-10° C.) and stirred at room temperature for 72 hours. The reactionmixture was evaporated and the resultant residue was purified by columnchromatography on silica gel eluting with dichloromethane/methanol(0-10%) to provide the title compound (0.093 g) as a white solid, afterwashing with ethyl acetate. M.p. 197°-199° C.; ¹ H NMR (400 MHz, Me₂SO-d₆) δ 3.57, 3.65 (2H, ABX, H-5'_(a) and H-5'_(b)), 3.92 (1H, q,H-4'), 4.12 (1H, m, H-3'), 4.50 (1H, q, H-2'), 5.80 (1H, d, H-1'), 8.35(1H, s, H-8). HPLC retention time 10.12 (gradient elution, 5-25%acetonitrile/0.1M pH 3.3 ammonium sulphate buffer: 214 nm detector):100% purity.

C₁₅ H₂₁ FN₆ O₄. O.2 EtOAc requires C, 49.2; H, 5.9; N, 21.8%. Found: C,49.2; H, 5.9; N, 21.75%.

EXAMPLE 10 (Method C) 2-Bromo-N-(1-piperidinyl)adenosine

9-(2',3',5'-Tri-O-acetyl-β-D-ribofuranosyl)-2-amino-6-chloro-9H-purine(2.2 g, 5.13 mmol) (prepared as described in example 9) was dissolved inacetonitrile (40 ml).

Bromoform (5.5 ml, 62.9 mmol) (dried by passage through an aluminacolumn) and 3-methylbutylnitrite (6.1 ml, 45.6 mmol) were introduced andthe reaction mixture was saturated with nitrogen before being heated at45° C. for 18 hours and allowed to cool to room temperature; the producthad r_(f) 0.47 (SiO₂, ethyl acetate). The reaction mixture wasevaporated in vacuo and purified by flash chromatography on silica gel;elution initially with dichloromethane followed bydichloromethane/methanol (50:1) provided the product which was dissolvedin a mixture of dichloromethane (2 ml) and ethanol (30 ml). Thedichloromethane was removed in vacuo and9-(2',3',5'-tri-O-acetyl-β-D-ribofuranosyl)-2-bromo-6-chloro-9H-purinecrystallized as a solid (2.11 g, 42%): m.p. 160°-162° C.

To a sample of9-(2',3',5'-tri-O-acetyl-β-D-ribofuranosyl)-2-bromo-6-chloro-9H-purine(1.0 g, 2.03 mmol) in dioxan (20 ml) 1-aminopiperidine (0.24 ml, 2.23mmol) was added followed by triethylamine (2.33 ml, 18.5 mmol) and thesolution was stirred for 20 hours at room temperature. The reactionmixture was filtered, evaporated and the resultant residue was purifiedby flash chromatography on silica gel. Elution initially withdichloromethane and then with dichloromethane/methanol (100:1) providedimpure 2',3',5'-tri-O-acetyl-2-bromo-N-(1-piperidinyl)adenosine whichwas repurified by flash chromatography in cyclohexane/ethyl acetate(1:1), giving the pure product as a foam (0.86 g, 76%).

The above 2',3',5'-tri-O-acetyl-2-bromo-N-(1-piperidinyl)adenosine wastreated with methanolic ammonia (10 ml) (previously saturated at -10°C.) and stirred at room temperature for 16 hours after which time TLCinvestigation indicated that the starting material was consumed and anew product was present [r_(f) 0.24 (SiO₂, ethyl acetate/methanol(90/10)]. The reaction mixture was evaporated and the resultant residuewas treated with water (25 ml) and the suspension was extracted withethyl acetate (2×25 ml). The combined extracts were dried (MgSO₄) andcoevaporated with dichloromethane; ¹ H NMR (400 MHz, Me₂ SO-d₆) δ 3.54,3.65 (2H, ABX, H-5'_(a) and H-5'_(b)), 3.94 (1H, q, H-4'), 4.12 (1H, m,H-3'), 4.50 (1H, m, H-2'), 5.82 (1H, d, H-1'), 8.38 (1H, s, H-8). HPLCretention time 14.25 (gradient elution, 5-25% acetonitrile/0.1M pH 3.3ammonium sulphate buffer: 214 nm detector).

C₁₅ H₂₁ BrN₆ O₄. O.33 CH₂ Cl₂. 0.75 H₂ O requires C, 40.1; H, 5.1; N,18.3%. Found: C, 40.5; H, 5.2; N, 17.8%.

EXAMPLE 11 (Method C) 2-Iodo-N-(1-pyrrolidinyl)adenosine

9-(2',3',5'-Tri-O-acetyl-β-D-ribofuranosyl)-2-amino-6-chloro-9H-purine(2.2 g, 5.13 mmol) (prepared as described in Example 9) (0.54 g, 1.0mmol) was dissolved in dioxan (5 ml). 1-Aminopyrrolidine hydrochloride(0.135 g, 1.1 mmol) was added followed by triethylamine (0.312 ml, 2.3mmol) and the solution was stirred for 72 hours at room temperature.Further 1-aminopyrrolidine hydrochloride (0.405 g, 3.3 mmol) andtriethylamine (0.935 ml, 3.45 mmol) were introduced and stirring wascontinued at room temperature for 24 hours. Ethanol (1 ml) was added andthe solution was heated at 50° C. for 2 hours. The reaction mixture wasevaporated and purified by column chromatography on silica gel elutinginitially with n-heptane/THF (4/1) and later with n-heptane/THF (1/1) toprovide9-(2',3',5'-tri-O-acetyl-β-D-ribofuranosyl)-2-iodo-6-(1-pyrrolidinyl)-(9H)-purine(0.14 g, 23%) as a solid which was recrystallized from ethanol giving0.08 g (14%); ¹ H NMR (400 MHz, Me₂ SO-d₆) δ 2.04, 2.07 (6H, s, 2'- and3'-O-acetyl-CH₃), 2.13 (3H, s, 5'-O-acetyl-CH₃) , 4.28 (1H, m,H-5'_(a)), 4.35-4.43 (2H, m, H-5'_(b) and H-4'), 6.15 (1H, d, H-1'),8.29 (1H, s, H-8).

The above9-(2',3',5'-tri-O-acetyl-β-D-ribofuranosyl)-2-iodo-6-(1-pyrrolidinyl)-(9H)-purine(0.063 g, 0.107 mmol) was suspended in methanol (1 ml) and driedpotassium carbonate (0.030 g, 0.22 mmol) was introduced. The reactionmixture was stirred for 24 hours at room temperature and furtherportions of methanol (10 ml) and dried potassium carbonate (0.030 g,0.22 mmol) were added. After a further 24 hours, the solution wastreated with glacial acetic acid (0.1 ml) and evaporated. The residuewas dissolved in a mixture of water (20 ml) and methanol (10 ml) and themethanol was removed by azeotropic distillation, causing the product tocrystallize. The title compound was obtained as white crystals, m.p.216°-217° C.; ¹ H NMR (400 MHz, Me₂ SO-d₆) δ 3.94 (1H, q, H-4'), 4.12(1H, q, H-3'), 4.50 (1H, q, H-2'), 5.83 (1H, d, H-1'), 8.28 (1H, s,H-8); HPLC retention time 13.70 (gradient elution, 25-45%acetonitrile/0.1M Ph 3.3 ammonium sulphate buffer: 214 nm detector):purity 97.5%.

EXAMPLE 12 (Method B) N-(1-Piperidinyl)-2-(1-propoxy)adenosine

2-Chloro-N-(1-piperidinyl)adenosine (0.50 g, 1.3 mmol) (Example 5) wasdissolved in a solution of sodium hydroxide (0.51 g, 1.3 mmol) in1-propanol (15 ml), and the solution was heated at reflux for 4.5 hours,after which time TLC investigation showed that the starting material hadbeen consumed. The reaction mixture was evaporated and the resultantresidue was dissolved in water. The solution was neutralized with 4Naqueous hydrochloric acid and extracted with dichloromethane (3×50 ml).The combined extracts were dried (MgSO₄) and evaporated to an oil whichwas triturated with diethyl ether to afford a fawn foam. This foam waspurified by column chromatography on silica gel (2×20 cm); elution witha mixture of dichloromethane/ethanol 25% aqueous ammonia solution(60/10/1) provided the title compound (0.20 g, 37%) as a semi-solidfoam; ¹ H NMR (400 MHz, Me₂ SO-d₆) δ 3.54, 3.65 (2H, ABX, H-5'_(a) andH-5'_(b)), 3.92 (1H, q, H-4'), 4.15 (1H, q, H-3'), 4.60 (1H, q, H-2'),5.80 (1H, d, H-1'), 8.16 (1H, s, H-8).

EXAMPLE 13 2-Chloro-N-(4-phenyl-1-piperazinyl)adenosine

The title compound was prepared according to method A as described inExample 6 by reacting 1-amino-4-phenylpiperazine hydrochloride (alsoprepared using the method described by Overberger, C. G. and Herin, L.P., Journal of Organic Chemistry, 1962, 27, 417) (0.77 g, 3.0 mmol) with9-(2',3',5'-tri-O-benzoyl-β-D-ribofuranosyl)-2,6-dichloro-9H-purine(1.90 g, 3.0 mmol), followed by debenzoylation of the purified productusing methanolic ammonia. This provided the title2-chloro-N-(4-phenyl-1-piperazinyl)adenosine (0.81 g, 60%) (after columnchromatography as a foam, ¹ H NMR (DMSO-d₆) δ 3.53-3.60 (1H, m,H-5'_(a)), 3.63-3.70 (1H, m, H-5'_(b)), 3.95 (1H, q, H-4'), 4.13 (1H, q,H-3'), 4.51 (1H, q, H-2'), 5.07 (1H, t, 5'-0H), 5.22, 5.50 (2H, 2d, 2'-and 3'-OH), 5.85 (1H, d, H-1'), 6.77-7.26 (5H, 3m, Ar--H), 8.44 (1H, s,H-8), 9.50 (1H, s, N--H).

C₂₀ H₂₄ ClN₇ O₄. H₂ O requires C, 50.1; H, 5.3; N, 20.4%. Found: C,50.2; H, 5.4; N, 20.0%.

EXAMPLE 14 2-Chloro-N-(4-phenyl-1,2,3,6-tetrahydro-1-pyridinyl)adenosine

The title compound was prepared according to method A as described aboveby reacting crude 1-amino-4-phenyl-1,2,3,6-tetrahydropyridine (preparedby the procedure outlined in Example 6) (1.25 g) with9-(2',3',5'-tri-O-benzoyl-β-D-ribofuranosyl)-2,6-dichloro-9H-purine(2.50 g, 3.9 mmol), followed by debenzoylation of the purified productusing methanolic ammonia to provide the title2-Chloro-N-(4-phenyl-1,2,3,6-tetrahydro-1-pyridinyl)adenosine (0.20 g12%) (after column chromatography) as a foam, ¹ H NMR (DMSO-d₆) δ3.53-3.70 (4H, m, H-5'_(a), H-5'_(b) and --CH₂ --), 3.94 (1H, q, H-4'),4.14 (1H, q, H-3'), 4.52 (1H, q, H-2'), 5.07 (1H, t, 5'-0H), 5.22, 5.50(2H, 2d, 2'- and 3'-OH), 5.85 (1H, d, H-1'), 6.15 (1H, br s, vinylicC--H), 7.24-7.51 (5H, m, Ar--H), 8.43 (1H, s, H-8), 9.55 (1H, s, N--H).

C₂₁ H₂₃ ClN₆ O₄, 1.25H₂ O requires C, 52.4; H, 5.3; N, 17.5%. Found: C,52.6; H, 5.0; N, 17.1%.

EXAMPLE 15 2-Chloro-N-(4-phenyl-1-piperidinyl)adenosine

The title compound was prepared according to method A as described aboveby reacting 1-amino-4-phenylpiperidine (prepared as outlined in Example6) (0.77 g, 3.6 mmol) with9-(2',3',5'-tri-O-benzoyl-β-D-ribofuranosyl)-2,6-dichloro-9H-purine(1.90 g, 3.0 mmol), followed by debenzoylation of the purified productusing methanolic ammonia. This provided the title2-chloro-N-(4-phenyl-1-piperidinyl)adenosine (0.49 g, 37%) as a solidwhich precipitated on evaporation of column chromatography fractions, mp142°-149° C. ¹ H NMR (DMSO-d₆) δ 3.53-3.60 (1H, m, H-5'_(a)), 3.63-3.70(1H, m, H-5'_(b)), 3.95 (1H, q, H-4'), 4.13 (1H, q, H-3'), 4.51 (1H, q,H-2'), 5.07 (1H, t, 5'-0H), 5.22, 5.50 (2H, 2d, 2'- and 3'-OH), 5.84(1H, d, H-1'), 7.18-7.35 (5H, 2m, Ar--H), 8.43 (1H, s, H-8) 9.45 (1H, s,N--H).

C₂₁ H₂₅ ClN₆ O₄ requires C, 54.6; H, 5.8; N, 17.4%. Found: C, 54.4; H,5.8; N, 17.0%.

EXAMPLE 16 2-Chloro-N-(3-phenoxy-1-piperidinyl)adenosine

1-(1,1-Dimethylethoxycarbonyl)-3-hydroxypiperidine

3-Hydroxypiperidine (10.1 g, 0.1 mol) was dissolved in tetrahydrofuran(50 ml) and 1N aqueous sodium hydroxide solution (95 ml) was introduced.The solution was cooled to 0° C. and a solution of di-tert-butyldicarbonate (24.0 g, 0.11 mol) in tetrahydrofuran was added over 1 h.The reaction mixture was stirred at ambient temperature for 18 h andevaporated to an aqueous suspension. Water (100 ml) was added and themixture was extracted with dichloromethane (3×100 ml). The combinedextracts were dried (MgSO₄), evaporated and the residue wasrecrystallised from n-heptane to provide the title product as a solid(15.71 g, 78%), mp 67°-69° C.

3-Phenoxypiperidine

1-(1,1-Dimethylethoxycarbonyl)-3-hydroxypiperidine (6.0 g, 30 mmol) wasdissolved in toluene (75 ml) and phenol (2.82 g, 30 mmol) was addedfollowed by triphenylphosphine (11.8 g, 45 mmol). To this mixture asolution of diethylazodicarboxylate (7.84 g, 45 mmol) in toluene (75 ml)was introduced dropwise and the reaction mixture was stirred for 18 h.at ambient temperature during which time triphenylphosphine oxideprecipitated. The reaction mixture was filtered, washed with 0.1N sodiumhydroxide solution (55 ml), 0.5N sodium bicarbonate (100 ml) and with amixture of saturated brine (25 ml) and water (25 ml). The dried toluenesolution was evaporated, the residue was dissolved in ethyl acetate (10ml) and cyclohexane (200 ml) was added. The solid precipitate wasremoved and the residue on evaporation was purified by flashchromatography on silica gel (7.5×15 cm). Elution with heptane ethylacetate (9/1) provided the phenyl ether as an oil (4.01 g, 40%),containing the desired intermediate as well as ca. 20% phenol. This oilwas dissolved in dichloromethane (15 ml) and trifluoroacetic acid (3 ml)was added. The solution was stirred at room temperature for 6 h and at-18° C. for 72 h. Saturated sodium bicarbonate solution was added to thereaction mixture until neutrality was reached, followed by extractionwith dichloromethane (6×30 ml). The combined extracts were dried (MgSO₄)and evaporated to an oil which was dissolved in toluene (200 ml)containing methanol (1.26 ml, 30 mmol). Chlorotrimethylsilane (1.82 ml,15 mmol) was added, and in the absence of crystallization, the solutionwas evaporated to an oil, which was treated with diethyl ether. Thesolid which was formed was dried in vacuo to give the title compound(1.90 g, 27%), mp 156°-159° C.

This 3-phenoxypiperidine was N-aminated using the method described inExample 6.

2-Chloro-N-(3-phenoxy-1-piperidinyl)adenosine was prepared according tomethod A as described in Example 6 by reacting1-amino-3-phenoxypiperidine (0.60 g, 3.4 mmol) with9-(2',3',5'-tri-O-benzoyl-β-D-ribofuranosyl)-2,6-dichloro-9H-purine (2.0g, 3.2 mmol), followed by debenzoylation of the purified product usingmethanolic ammonia. This provided the desired compound (0.65 g, 43%)(after column chromatography) as a foam, ¹ H NMR (DMSO-d₆) δ 3.53-3.60(1H, m, H-5'_(a)), 3.64-3.72 (1H, m, H-5'_(b)), 3.96 (1H, q, H-4'), 4.14(1H, q, H-3'), 4.50-4.59 (1H, m, H-2' and --C--H) ), 5.07 (1H, t,5'-0H), 5.23, 5.50 (2H, 2d, 2'- and 3'-OH), 5.84 (1H, d, H-1'),6.79-7.32 (5H, 3m, Ar--H), 8.45 (1H, s, H-8), 9.55 (1H, s, N--H).

C₂₁ H₂₅ ClN₆ O₅. 0.5 H₂ O requires C, 51.9; H, 5.4; N, 17.3%. Found: C,51.9; H, 5.5; N, 17.1%.

EXAMPLE 17 2-Chloro-N-[4-phenoxy-1-piperidinyl]adenosine

This compound was prepared from 4-hydroxypiperidine using themethodology described in Example 16. 1-Amino-4-phenoxypiperidine (0.96g, 5.0 mmol) was reacted with9-(2',3',5'-tri-O-benzoyl-β-D-ribofuranosyl)-2,6-dichloro-9H-purine(2.50 g, 4 mmol), followed by debenzoylation of the purified productusing methanolic ammonia. This provided the title2-chloro-N-(4-phenoxy-1-piperidinyl)adenosine (1.07 g, 57%) as a foam, ¹H NMR (DMSO-d₆) δ 3.52-3.60 (1H, m, H-5'_(a)), 3.63-3.70 (1H, m,H-5'_(b)), 3.95 (1H, q, H-4'), 4.14 (1H, q, H-3'), 4.43-4.54 (2H, m,H-2' and --C--H), 5.07 (1H, t, 5'-0H), 5.22, 5.50 (2H, 2d, 2'- and3'-OH), 5.84 (1H, d, H-1'), 6.90-7.33 (5H, 2m, Ar--H), 8.42 (1H, s,H-8), 9.49 (1H, s, N--H).

C₂₁ H₂₅ ClN₆ O₄. H₂ O requires C, 51.0; H, 5.5; N, 17.0%. Found: C,50.9; H, 5.2; N, 16.6%.

EXAMPLE 18 2-Chloro-N-(3-phenylthio-1-piperidinyl)adenosine

3-Phenylthiopiperidine was prepared from1-(1,1-dimethylethoxycarbonyl)-3-hydroxypiperidine by the methoddescribed by Kotsuki et al., Tetrahedron Letters, 1991, 32, 4155-4158;otherwise the synthesis proceeded as described in Example 16.1-Amino-3-phenylthiopiperidine (0.98 g, 4.0 mmol) was reacted with9-(2',3',5'tri-O-benzoyl-β-D-ribofuranosyl)2,6-dichloro-9H-purine (2.11g, 3.3 mmol), followed by debenzoylation of the purified product usingmethanolic ammonia. This provided the title2-chloro-N-(3-phenylthio-1-piperidinyl)adenosine (0.89 g, 55%) as afoam, ¹ H NMR (DMSO-d₆) δ 3.52-3.59 (1H, m, H-5'_(a)), 3.63-3.71 (1H, m,H-5'_(b)) 3.95 (1H, q, H-4'), 4.13 (1H, q, H-3'), 4.46-4.54 (1H, q,H-2'), 5.07 (1H, t, 5'-0H), 5.22, 5.49 (2H, 2d, 2'- and 3'-OH), 5.84(1H, d, H-1'), 7.20-7.50 (5H, 2m, Ar--H), 8.43 (1H, s, H-8), 9.50 (1H,s, N--H).

C₂₁ H₂₅ ClN₆ O₄, 0.5 H₂ O requires C, 50.2; H, 5.2; N, 16.7%. Found: C,50.0; H, 5.3; N, 16.6%.

EXAMPLE 19 2-Chloro-N-(4-phenylthio-1-piperidinyl)adenosine

The title compound was prepared as described in Example 18.

1-Amino-4-phenylthiopiperidine (1.10 g, 6.7 mmol) was reacted with9-(2',3',5'tri-O-benzoyl-β-D-ribofuranosyl)-2,6-dichloro-9H-purine (2.5g, 4 mmol), followed by debenzoylation of the purified product usingmethanolic ammonia. This provided the title2-chloro-N-(4-phenylthio-1-piperidinyl)adenosine (1.25 g, 65%) as afoam, ¹ H NMR (DMSO-d₆) δ 3.51-3.60 (1H, m, H-5'_(a)), 3.62-3.68 (1H, m,H-5'_(b)), 3.95 (1H, q, H-4'), 4.14 (1H, q, H-3'), 4.50 (1H, q, H-2'),5.08 (1H, t, 5'-0H), 5.21, 5.50 (2H, 2d, 2'- and 3'-OH), 5.83 (1H, d,H-1'), 7.24-7.45 (5H, 2m, Ar--H), 8.41 (1H, s, H-8), 9.44 (1H, s, N--H).

C₂₁ H₂₅ ClN₆ O₄ S, 0.75 H₂ O requires C, 49.8; H, 5.3; N, 16.6%. Found:C, 49.6; H, 5.2; N, 16.5%.

EXAMPLE 20 2-Chloro-N-[2-(phenylthiomethyl)-1-piperidinyl]adenosine

2-(Phenylthiomethyl)piperidine was prepared from2-(hydroxymethyl)piperidine by the method described by Kotsuki et al.,Tetrahedron Letters, 1991, 32, 4155-4158; otherwise the synthesisproceeded as described in Example 16.1-Amino-2-(phenylthiomethyl)piperidine (1.4 g, 6.5 mmol) was reactedwith 9-(2',3',5'tri-O-benzoyl-β-D-ribofuranosyl)-2,6-dichloro-9H-purine(2.0 g, 3.25 mmol), followed by debenzoylation of the purified productusing methanolic ammonia. This provided the title2-chloro-N-[2-(phenylthiomethyl)-1-piperidinyl]adenosine (0.17 g, 10%)as a foam (a mixture of diastereoisomers); ¹ H NMR (DMSO-d₆) δ 3.51-3.59(1H, m, H-5'_(a)), 3.62-3.70 (1H, m, H-5'_(b)), 3.95 (1H, q, H-4'), 4.13(1H, q, H-3'), 4.47-4.56 (1H, m, H-2'), 5.06 (1H, t, 5'-0H), 5.22, 5.50(2H, 2d, 2'- and 3'-OH), 5.82-5.87 (1H, 2d, H-1'), 7.16-7.54 (5H, 2m,Ar--H), 8.41, 8.46 (1H, 2s, H-8), 9.40 (1H, s, N--H).

EXAMPLE 21 2-Chloro-N-(3-hydroxypiperidinyl)adenosine

The title compound was prepared according to method A as described aboveby reacting 1-amino-3-hydroxypiperidine (prepared from3-hydroxypiperidine as outlined in Example 6) (0.60 g, 5.9 mmol) with9-(2',3',5'-tri-O-benzoyl-β-D-ribofuranosyl)-2,6-dichloro-9H-purine(2.50 g, 3.94 mmol), followed by debenzoylation of the purified productusing methanolic ammonia to provide the title2-chloro-N-(3-hydroxypiperidinyl) adenosine (0.12 g, 8%) (after columnchromatography) as a foam (a mixture of diastereoisomers); ¹ H NMR(DMSO-d₆) δ 3.52-3.60 (1H, m, H-5'_(a)), 3.63-3.70 (1H, m, H-5'_(b)),3.95 (1H, q, H-4'), 4.14 (1H, m, H-3'), 4.52 (1H, m, H-2'), 5.08 (1H, t,5'-0H), 5.22, 5.50 (2H, 2d, 2'- and 3'-OH), 5.84 (1H, d, H-1'), 8.43(1H, br s, H-8), 9.45 (1H, 2 br s, N--H).

EXAMPLE 22 2-Chloro-N-(4-phenylsulphonyl-1-piperidinyl)adenosine

2-Chloro-N-(4-phenylthio-1-piperidinyl)adenosine (Example 19) (0.25 g,0.5 mmol) was dissolved in methanol (2 ml) and a solution of potassiumhydrogen persulphate ("Oxone" ) (Trost, B. M. and Curran, D. P.,Tetrahedron Letters, 1981, 22, 1287-1290) (0.47 g, 0.76 mmol) in water(2 ml) was added at 0° C. The yellowish reaction mixture was stirred atambient temperature for 4 h., and saturated sodium bicarbonate (10 ml)was introduced. The suspension was extracted with dichloromethane (2×50ml), and a yellow gum was seen to appear in the aqueous phase. Thisyellow gum was dissolved in methanol (20 ml), the dried dichloromethaneextracts were added, and the mixture was evaporated to a residue.Purification by "flash" chromatography, eluting initially withdichloromethane, proceeding to dichloromethane/methanol (9/1) providedthe title nucleoside as a foam (0.04 g, 15%), ¹ H NMR (DMSO-d₆) δ3.50-3.60 (1H, m, H-5'_(a)), 3.62-3.68 (1H, m, H-5'_(b)), 3.93 (1H, brq, H-4'), 4.12 (1H, m, H-3'), 4.50 (1H, m, H-2'), 5.05 (1H, t, 5'-0H),5.21, 5.48 (2H, 2d, 2'- and 3'-OH), 5.82 (1H, d, H-1'), 7.66-7.94 (5H,2m, Ar--H), 8.41 (1H, s, H-8), 9.49 (1H, s, N--H).

EXAMPLE 23 (Method C) 2-Methylthio-N-(1-piperidinyl)adenosine

9-(2',3',5'-Tri-O-acetyl-β-D-ribofuranosyl)-6-chloro-2-methylthio-9H-purine

9-(2',3',5'-Tri-O-acetyl-β-D-ribofuranosyl)-2-amino-6-chloro-9H-purine(see Example 9) (4.0 g, 9.3 mmol) was dissolved in acetonitrile (100ml). Isoamylnitrite (10.84 g, 93 mmol) was introduced followed by methyldisulphide (4.14 ml, 46 mmol) and the reaction mixture was heated at anoil bath temperature of 100° C. for 2h. The evolved gas was removed viaa hypochlorite scrubber. The reaction mixture was cooled, evaporated andpurified by flash chromatography on silica gel. Elution initially withdichloromethane, followed by dichloro-methane/methanol (100/1) provided9-(2',3',5'-tri-O-acetyl-β-D-ribofuranosyl)-6-chloro-2-methylthio-9H-purine(3.1 g, 72%) as a foam, ¹ H NMR (CDCl₃) δ 2.12, 2.14, 2.18 (9H, 3s, 2',3' and 5'-O-acetyl CH₃), 2.66 (3H, s, --SCH₃), 4.28-4.51 (3H, m,H-5'_(a), H-5'_(b) and H-4'), 5.66 (1H, t, H-3'), 6.0 (1H, t, H-2'),6.13 (1H, d, H-1'), 8.11 (1H, s, H-8).

The above9-(2',3',5'-tri-O-acetyl-β-D-ribofuranosyl)-6-chloro-2-methylthio-9H-purine(1.83 g, 3.9 mmol) was dissolved in dioxan (40 ml) followed by1-aminopiperidine (0.59 g, 5.85 mmol) and triethyl-amine (1.63 ml, 11.7mmol). The reaction mixture was stirred at ambient temperature for 18 h,evaporated and purified by flash chromatography on silica gel. Elutioninitially with dichloromethane, followed by dichloromethane/methanol(100/1) provided9-(2',3',5'-tri-O-acetyl-β-D-ribofuranosyl)-2-methylthio-6-(1-piperidinyl)-9H-purine(1.24 g, 59%) as a foam. This compound was dissolved in methanolicammonia (10 ml) and the solution was stirred at room temperature for 18h, evaporated and purified by flash chromatography on silica gel.Elution initially with dichloromethane, followed bydichloromethane/methanol (19/1) gave 2-methylthio-N-(1-piperidinyl)adenosine (0.67 g, 55%) as a foam, ¹ H NMR (DMSO-d₆) δ 2.51 (3H, s,--SCH₃), 3.50-3.56 (1H, m, H-5'_(a)), 3.62-3.68 (1H, m, H-5'_(b)) 3.92(1H, q, H-4'), 4.15 (1H, q, H-3'), 4.50 (1H, q, H-2'), 5.03 (1H, t,5'-0H), 5.18, 5.44 (2H, 2d, 2'- and 3'-OH), 5.84 (1H, d, H-1'), 8.24(1H, s, H-8), 8.84 (1H, s, N--H).

C₁₆ H₂₄ N₆ O₄ S.H₂ O requires C, 46.4; H, 6.3; N, 20.3%. Found: C, 46.1;H, 6.0; N, 19.8%.

We claim:
 1. A method of treating myocardial or cerebral ischemia,comprising administering to a person in need thereof an effective amountof a compound of formula I: ##STR9## wherein X is halogen,perhalomethyl, cyano, C₁₋₆ -alkoxy, C₁₋₆ -alkylthio or C₁₋₆-alkylamino;R¹ is ##STR10## which is optionally substituted with one ortwo C₁₋₆ -alkyl groups, C₂₋₆ -alkenyl, C₂₋₆ -alkynyl, phenoxy,phenylsulphonyl, phenylthio, hydroxy, phenyl, C₁₋₆ -alkoxy or -C₁₋₆-alkyl-C₁₋₆ -alkoxy, wherein n is 1; or a pharmaceutically acceptablesalt thereof.
 2. The method according to claim 1, wherein the compoundis selected from the group consistingof(R)-2-Chloro-N-[2-(methoxymethyl)-1-pyrrolidinyl]adenosine;(S)-2-Chloro-N-[2-(methoxymethyl)-1-pyrrolidinyl]adenosine;2-Iodo-N-(1-pyrrolidinyl)adenosine; andpharmaceutically acceptable saltsthereof.
 3. A method of wearing myocardial or cerebral ischemia,comprising administering to a person in need thereof an effective amountof a compound of formula I: ##STR11## wherein X is halogen,perhalomethyl, cyano, C₁₋₆ -alkoxy or C₁₋₆ -alkylthio;R¹ is ##STR12##which is optionally substituted with one or two C₁₋₆ -alkyl groups, C₂₋₆-alkenyl, C₂₋₆ -alkynyl, phenoxy, phenylsulphonyl, phenylthio, hydroxy,phenyl, C₁₋₆ -alkoxy, -C₁₋₆ -alkyl-C₁₋₆ -alkoxy or phenylthiomethyl,wherein n is 2; or a pharmaceutically acceptable salt thereof.
 4. Themethod according to claim 3, wherein X is halogen.
 5. The methodaccording to claim 3, wherein R¹ is unsubstituted or substituted withphenoxy.
 6. The method according to claim 3, wherein the compound isselected from the group consistingof2-Chloro-N-(2,6-dimethyl-1-piperidinyl)adenosine;2-Chloro-N-(1-piperidinyl)adenosine;2-Fluoro-N-(1-piperidinyl)adenosine; 2-Bromo-N-(1-piperidinyl)adenosine;2-Chloro-N-(4-phenyl-1-piperidinyl)adenosine;2-Chloro-N-(3-phenoxy-1-piperidinyl)adenosine;2-Chloro-N-(4-phenoxy-1-piperidinyl)adenosine;2-Chloro-N-(3-phenylthio-1-piperidinyl)adenosine;2-Chloro-N-(4-phenylthio-1-piperidinyl)adenosine;2-Chloro-N-(3-hydroxypiperidinyl)adenosine;2-Chloro-N-(4-phenylsulphonyl-1-piperidinyl)adenosine;andpharmaceutically acceptable salts thereof.
 7. The method according toclaim 3, wherein the compound is selected from the group consistingof2-Chloro-N-(2-phenyl-1-piperidinyl)adenosine;N-(1-Piperidinyl)-2-(1-propoxy)adenosine;2-Chloro-N-[2-(phenylthiomethyl)-1-piperidinyl]adenosine;2-Methylthio-N-(1-piperidinyl)adenosine; andpharmaceutically acceptablesalts thereof.
 8. A method of treating myocardial or cerebral ischemia,comprising administering to a person in need thereof an effective amountof a compound of formula I: ##STR13## wherein X is halogen,perhalomethyl, cyano, C₁₋₆ -alkoxy, C₁₋₆ -alkylthio or C₁₋₆-alkylamino;R¹ is ##STR14## which is optionally substituted with one ortwo C₁₋₆ -alkyl groups, C₂₋₆ -alkenyl, C₂₋₆ -alkynyl, phenoxy,phenylsulphonyl, phenylthio, hydroxy, phenyl, C₁₋₆ -alkoxy or -C₁₋₆-alkyl-C₁₋₆ -alkoxy, wherein n is 3; ora pharmaceutically acceptablesalt thereof.
 9. The method according to claim 8, wherein the compoundis2-Chloro-N-[1-(2,3,4,5,6,7-hexahydro)azepinyl]adenosine or apharmaceutically acceptable salt thereof.
 10. A method of treatingmyocardial or cerebral ischemia, comprising administering to a person inneed thereof an effective amount of a compound of formula I: ##STR15##wherein X is halogen, perhalomethyl, cyano, C₁₋₆ -alkoxy, C₁₋₆-alkylthio or C₁₋₆ -alkylamino;R¹ is ##STR16## which is optionallysubstituted with C₁₋₆ -alkyl, C₂₋₆ -alkenyl, C₂₋₆ -alkynyl, phenoxy,phenyl, C₁₋₆ -alkoxy or -C₁₋₆ -alkyl-C₁₋₆ -alkoxy, wherein Y is O; orapharmaceutically acceptable salt thereof.
 11. The method according toclaim 10, wherein the compound is2-Chloro-N-(4-morpholinyl)adenosine ora pharmaceutically acceptable salt thereof.
 12. A method of treatingmyocardial or cerebral ischemia, comprising administering to a person inneed thereof an effective amount of a compound of formula I: ##STR17##wherein X is halogen, perhalomethyl, cyano, C₁₋₆ -alkoxy, C₁₋₆-alkylthio or C₁₋₆ -alkylamino;R¹ is ##STR18## which is optionallysubstituted with C₁₋₆ -alkyl, C₂₋₆ -alkenyl, C₂₋₆ -alkynyl, phenoxy,phenyl, C₁₋₆ -alkoxy or -C₁₋₆ -alkyl-C₁₋₆ -alkoxy, wherein Y is S; orapharmaceutically acceptable salt thereof.
 13. A method of treatingmyocardial or cerebral ischemia, comprising administering to a person inneed thereof an effective amount of a compound of formula I: ##STR19##wherein X is halogen, perhalomethyl, cyano, C₁₋₆ -alkoxy, C₁₋₆-alkylthio or C₁₋₆ -alkylamino;R¹ is ##STR20## which is optionallysubstituted with C₁₋₆ -alkyl, C₂₋₆ -alkenyl, C₂₋₆ -alkynyl, phenoxy,phenyl, C₁₋₆ -alkoxy or -C₁₋₆ -alkyl-C₁₋₆ -alkoxy, wherein Y is NZwherein Z is H, C₁₋₆ -alkyl or phenyl; ora pharmaceutically acceptablesalt thereof.
 14. The method according to claim 13, wherein the compoundis selected from the group consistingof2-Chloro-N-(4-methyl-1-piperazinyl)adenosine;2-Chloro-N-(4-phenyl-1-piperazinyl)adenosine; andpharmaceuticallyacceptable salts thereof.
 15. A method of treating myocardial orcerebral ischemia, comprising administering to a person in need thereofan effective amount of a compound of formula I: ##STR21## wherein X ishalogen, perhalomethyl, cyano, C₁₋₆ -alkoxy, C₁₋₆ -alkylthio or C₁₋₆-alkylamino;R¹ is ##STR22## which is optionally substituted with C₁₋₆-alkyl, C₂₋₆ -alkenyl, C₂₋₆ -alkynyl, phenoxy, phenylthio, phenyl, C₁₋₆-alkoxy or -C₁₋₆ -alkyl-C₁₋₆ -alkoxy; orpharmaceutically acceptable saltthereof.
 16. The method according to claim 15, wherein the compoundis2-Chloro-N-(4-phenyl-1,2,3,6-tetrahydro-1-pyridinyl)adenosine or apharmaceutically acceptable salt thereof.